Stabilizing Off-pathway Oligomers by Polyphenol Nanoassemblies for IAPP Aggregation Inhibition

Abstract

Experimental studies have shown that many naturally occurring polyphenols have inhibitory effect on the aggregation of several proteins. Here, we use discrete molecular dynamics (DMD) simulations and high-throughput dynamic light scattering (DLS) experiments to study the anti-aggregation effects of two polyphenols, curcumin and resveratrol, on the aggregation of islet amyloid polypeptide (IAPP or amylin). Our DMD simulations suggest that the aggregation inhibition is caused by stabilization of small molecular weight IAPP off-pathway oligomers by the polyphenols. Our analysis indicates that IAPP-polyphenol hydrogen bonds and π-π stacking combined with hydrophobic interactions are responsible for the stabilization of oligomers. The presence of small oligomers is confirmed with DLS measurements in which nanometer-sized oligomers are found to be stable for up to 7.5 hours, the time frame within which IAPP aggregates in the absence of polyphenols. Our study offers a general anti-aggregation mechanism for polyphenols, and further provides a computational framework for the future design of anti-amyloid aggregation therapeutics.

Figure 1. Interaction of small molecules with IAPP monomer.

(A–C) Representative snapshots showing the final structures from simulations with (A) aspirin, (B) curcumin and (C) resveratrol. (D) Contact frequency of small molecules with IAPP residues. The IAPP-small molecule contact is dominated by the hydrophobic residues. (D) Small molecules stabilize helices while the β-strand content of IAPP is reduced.

Figure 2. Interactions of small molecules with multiple IAPPs.

(A) IAPP-IAPP contact number as a function of number of IAPP chains. The contact number is smaller in the presence of curcumin and resveratrol, while aspirin has no apparent effect. (B) Secondary structure contents for the octamer IAPP system. The overall number of helical residues is increased in comparison with the monomer IAPP system. Also, the small molecules have an opposite effect on the helical and strand contents of IAPP. (C) Typical trajectories of potential energy and IAPP-curcumin contact number for the octamer IAPP system. Snapshots of the system at different times are shown in the inset of (C). Representative final snapshots of IAPP systems with (D) resveratrol, (E) aspirin and (F) no ligand.

Figure 3. Clustering properties of the octamer IAPP system.

(A) The probability of the number of IAPP-containing clusters. For IAPP alone and IAPP-aspirin systems one or two clusters are most populated while three clusters is most populated for IAPP-resveratrol and IAPP-curcumin systems. The error bars correspond to standard deviations from independence simulations. (B) Histogram of the cluster size. Three and five IAPP-containing clusters are most populated for curcumin- and resveratrol-IAPP systems, while eight IAPP-containing cluster is the most common one for IAPP alone and IAPP-aspirin systems. (C,D) Compositions heat map for IAPP-curcumin and IAPP-resveratrol clusters.

Figure 4. Properties of stable small molecule-IAPP clusters.

(A,B) Radial distributions of IAPP and small molecule atoms from the center of mass of the cluster, for (A) curcumin-IAPP and (B) resveratrol-IAPP clusters. Typical structures of the clusters are shown in inset; protein atoms in golden-yellow, curcumin in green (carbon) and red (oxygen), and resveratrol in blue (carbon) and red (oxygen). (C,D) Distributions of small aliphatic carbon and oxygen atoms of curcumin and resveratrol inside the clusters. (Inset) Structures of curcumin and resveratrol, highlighting the oxygen atoms used for calculations as spheres.

Figure 5. Time evolution of the hydrodynamic diameters of IAPP

(A) and IAPP-resveratrol at 1:2, 1:2.7 and 1.3.1 molar ratios (B–D). IAPP concentration in all samples: 19 μM.

Figure 6. Stabilizing forces of small molecule-IAPP clusters.

(A) Curcumin-IAPP and (B) resveratrol-IAPP clusters are stabilized by π-π stacking and hydrogen bonds. Zoomed-in snapshots depict stacking of phenyl groups of the small molecules against that of the protein. The hydroxyl groups of the small molecules make hydrogen bonds with the protein backbones and side chain atoms (shown using magenta dashed lines).